Interactions between binding molecules, which in general are biomolecules and their corresponding ligands, are central to life. Cells often bear or contain receptor molecules that interact or bind with a hormone, a peptide, a drug, an antigen, an effector molecule or with another receptor molecule; enzymes bind with their substrate; antibody molecules bind with an antigen, nucleic acid with protein, and so on. By “interact or bind” it is meant that the binding molecule and ligand approach each other within the range of molecular forces and may influence each other's properties. This approach takes the binding molecule and its ligand through various stages of molecular recognition comprising increasing degrees of intimacy and mutual effect: they bind.
Binding molecules have this binding ability because they comprise distinct binding sites allowing for the recognition of the ligand in question. The ligand, in turn, has a corresponding binding site, and only when the two binding sites can interact by—essentially spatial—complementarity, the two molecules can bind. Needless to say, molecules having three dimensions have binding sites that are of a three dimensional nature, often one or more surface projections or protuberances of one binding site correspond to one or more pockets or depressions in the other, a three-dimensional lock-and-key arrangement, sometimes in an induced-fit variety.
Sometimes, such a protuberance comprises a single loop of the molecule in question, and it is only this protuberance that essentially forms the binding site. In that case, one often terms these binding sites as comprising a linear or continuous binding site, wherein a mere linear part of the molecule in question is essentially responsible for the binding interaction. This terminology is widely used to describe, for example, antibody-antigen reactions wherein the antigen comprises part of a protein sequence, a linear peptide. One then often speaks about a linear or continuous epitope, wherein the binding site (epitope) of the antigenic molecule is formed by a loop of consecutively bound amino acids. However, similar continuous binding sites (the terms “epitope” and “binding site” are used interchangeably herein) can be found with receptor-antigen interactions (such as with a T-cell receptor), with receptor-ligand interactions such as with hormone receptors and agonists or antagonists thereof, with receptor-cytokine interactions, or with, for example, enzyme-substrate or receptor-drug interactions, whereby a linear part of the molecule is recognized as the binding site, and so on.
More often, however, such a protuberance or protuberances and depressions comprise various, distinct parts of the molecule in question, and the combined parts essentially form the binding site. Commonly, one names such a binding site comprising distinct parts of the molecule in question a discontinuous or conformational binding site or epitope. For example, binding sites laying on proteins having not only a primary structure (the amino acid sequence of the protein molecule), but also secondary and tertiary structure (the folding of the molecule into alpha-helices or beta-sheets and its overall shape), and sometimes even quaternary structure (the interaction with other protein molecules) may comprise in their essential protuberances or depressions amino acids or short peptide sequences that lay far apart in the primary structure but are folded closely together in the binding site.
Due to the central role binding molecules and their ligands play in life, there is an ever expanding interest in testing for or identification of the nature or characteristics of the binding site. Notably, the rapid developments in evolving biotechnology fields such as proteomics will result in the near future in the identification of more and more binding molecules and their corresponding ligands. The detection of protein-protein interactions and enzyme-substrate interactions (not only of protein enzymes but certainly also of for example catalytic RNA-based interactions), and the identification of protein-nucleic acid and of nucleic acid-nucleic acid pairs of binding molecule and corresponding ligand, will certainly result in generating more interest in where the exact interacting (binding) sites between these molecules lay and how one can develop compounds (agonists, antagonists, drugs) modulating the specific interaction.
Not only is one interested in the exact nature of the particular interaction between binding molecule and ligand in question, for example, in order to replace or supplement binding molecules or ligands when needed, but one is also interested in knowing approximating characteristics of the interaction in order to find or design analogues, agonists, antagonists or other compounds mimicking a binding site or ligand involved.
Versatile and rapid methods to test for or identify continuous epitopes or binding sites are known. Most, if not all, nucleic acid detection techniques, and molecular libraries using these, entail hybridization of an essentially continuous nucleic acid stretch with a complementary nucleic acid strand, be it DNA, RNA or PNA. Little attention has been paid to methods allowing rapid and straightforward identification of discontinuous binding sites of an essentially nucleic acid nature. Although plenty of such sites exist, think only of the lack of understanding surrounding ribosomal binding sites where ribosomal proteins bind to tRNA, of regulatory sites in promoter sequences, of interactions between polymerases and replicases between DNA and RNA, of catalytic RNA reactions, and so on, no molecular libraries exist that provide easy access to such sites.
An early work in the peptide field is disclosed in PCT International Publication No. WO 84/03564, related to a method of detecting or determining antigenically active amino acid sequences or peptides in a protein. This work, providing the so-called Pepscan technology, whereby a plurality of different peptides is synthesized by linking with a peptide bond a first amino acid to a second, and so on, and on a second position in the test format yet another first amino acid is linked to a second, and so on, after which the synthesized peptides are each tested with the binding molecule in question, allows the determination of every continuous antigenic determinant or continuous epitope of importance in a protein or peptide sequence. Pepscan technology taken in a broad sense also provides for the testing for or identification of (albeit linear) peptides essentially identical with, analogous to or mimicking binding sites or ligands of a various nature (mimotopes, Geyssen et al., Mol. Immunol. 23:709-715, 1986).
Pepscan technology allows identification of linear peptide sequences interacting with receptor molecules, enzymes, antibodies, and so on, in a rapid and straightforward fashion, allowing testing of a great many peptides for their reactivity with the binding molecule in question with relatively little effort. The order of magnitude of testing capability having been developed with Pepscan technology (e.g., also due to miniaturization of test formats; see, e.g., PCT International Publication No. WO 93/09872) furthermore allows at-random testing of a multiplicity of peptides, leading to automated combinatorial chemistry formats wherein a great many binding molecules are tested in a (if so desired at-random) pattern for their reactivity with a molecular library of synthetic peptides representing potential continuous binding sites or ligands, allowing the rapid detection of particularly relevant molecules out of tens of thousands of combinations of molecules tested.
However, for the testing of discontinuous or conformational binding sites to a binding molecule, no formats similar to or as versatile as Pepscan technology exist. Attempts to identify discontinuous epitopes by Pepscan technology are cumbersome. It does, in general, not suffice to merely extend synthesis of the test peptides by linking more amino acids to the existing peptide and hoping that some of the thus formed longer peptides will fold in such a way that at least two distinct parts are presented in a discontinuous fashion and are recognized by a binding molecule. In that case, there is no way of finding out in a rapid and straightforward fashion that the binding is indeed through a discontinuous binding site; it might be that just a longer single loop is responsible for the binding.
Some additional possibilities are provided by testing synthetic peptide sequences that have been designed to comprise two previously identified parts of a binding site, each part in essence being linear and being part of a larger linear peptide. Early work herein was done by Atassi and Zablocki (J. Biol. Chem 252:8784, 1977) who describe that spatially or conformationally contiguous surface residues (which are otherwise distant in sequence) of an antigenic site of egg white lysozyme were linked by peptide bonds into a single peptide which does not exist in lysozyme but attempts to simulate a surface region of it. However, their technique, called surface simulation synthesis, requires detailed knowledge of the three-dimensional structure of the protein under study and a full chemical identification of the residues constituting the binding site beforehand, as well as their accurate conformational spacing and directional requirements.
In the same fashion, Dimarchi et al. (Science 232:339-641, 1986) describe a 38 to 40 amino acid-long synthetic peptide consisting of two previously identified separate peptidyl regions of a virus coat protein. The peptide was synthesized using common peptide synthesis technology (Merrifield et al., Biochemistry 21, 5020, 1982) by adding subsequent amino acids with a peptide bond to an ever growing peptide resulting in a peptide wherein the two peptidyl regions were connected by a diproline spacer presumably functioning as indication of a secondary structural turn, thereby providing a two-part epitope or binding site.
However, it is clear that when one has to know beforehand the sequence of the (in this case only) two relevant parts in order to provide the desired discontinuous binding site, it excludes the feasibility of providing (desirably in a random fashion) a whole array of merely potential discontinuous binding sites for large scale testing. Furthermore, a major drawback of the above-mentioned strategies is that, again, only linear epitopes or dominant binding regions of discontinuous epitopes can be mimicked adequately. For the more complete synthesis of a discontinuous binding site, all the contributing parts have to be arranged in the proper conformation to achieve high-affinity binding. Therefore, single parts of discontinuous binding sites have to be linked.
Fifteen years after Dimarchi, Reineke et al. (Nature Biotechnology, 17:271-275, 1999) provided a synthetic mimic of a discontinuous binding site on a cytokine and a method to find such a discontinuous binding site that allowed for some flexibility and somewhat larger scale testing, wherein positionally addressable peptide collections derived from two separate regions of the cytokine were displayed on continuous cellulose membranes and substituted in the process to find the best binding peptide. After selection of the “best reactors” from each region, these were combined to give rise to another synthetic peptide collection (comprising peptides named duotopes) that again underwent several rounds of substitutions.
Reineke et al. thus provide synthesis of peptide chains comprising duotopes, however, again selected after previous identification of putative constituting parts with Pepscan technology, thereby still not allowing testing discontinuous binding sites in a rapid and straight forward fashion.
However, as indicated before, protein domains or small molecules that mimic binding sites are playing an increasing role in drug discovery, diagnostics and biotechnology. The search for particular molecules that bind to a binding site and mimic or antagonize the action of a natural ligand has been initiated in many laboratories. As indicated before, attempts to find such structures in synthetic molecular libraries often fail because of the essentially discontinuous nature and spatial complementarity of most binding sites.
Thus, for the many more cases where the binding site may essentially be discontinuous, improved means and methods to identify these sites are needed, and, in particular, means and methods are needed that allow testing for discontinuous binding sites whereby said parts need not necessarily first be selected by previous identification as a putative or even only tentative constituting part of the desired discontinuous binding site but bear only the potentiality of being part of that site by being a molecule with more or less distinct features per se.